The goal of this research is to determine how the dynamics of the actin and microtubule cytoskeletons are coordinated by a group of three interacting mammalian proteins: APC, Dia1, and EB1. This work will define the functions and mechanisms of these proteins, and provide a deeper understanding of the activities and interactions that underlie such processes as cell migration and morphogenesis. This project uses bulk biochemical experiments combined with a novel multi-wavelength single molecule biophysics method tailored to elucidate the mechanisms of complex, multi-component regulatory systems in vitro. In addition, the mechanisms deduced from the experiments in vitro will be tested in vivo to verify that they are important for relevant biological functions of these proteins in living cells (e.g., directed cell migration). The Specific Aims are: (1) Test two key hypotheses about the mechanism by which Dia1 and APC synergize in promoting actin assembly, involving formation of proposed physical complexes among components; (2) Test the additional hypothesis that Dia1 and APC synergize to stimulate actin assembly by a rocket launcher mechanism; and (3) Define the mechanisms by which EB1 alone and EB1 plus microtubules regulate and/or organize APC/Dia-induced actin assembly.